This proposal will examine how the intestinal brush border (BB) Na+/H+ exchanger 3 (NHE3) is inhibited by elevated Ca2+. This elevated Ca2+ regulatory process is relevant to both digestive physiology (postprandial cholinergic effects) and pathophysiology (diarrhea caused by some enterotoxins, hormones and laxatives occurs by elevation of Ca2+). The hypothesis to be tested in this proposal is that in addition to the second messenger Ca2+, NHE3 regulation requires and specificity is determined by formation of NHE3 containing plasma membrane complexes. These complexes include NHE3, PDZ domain containing scaffolding proteins, plus multiple additional regulatory proteins. This hypothesis is supported by strong preliminary data that have identified (1) E3KARP (NHE3 kinase a regulatory protein) as one PDZ domain containing protein involved and (2) (-actinin-4 and (3) protein kinase Calpha as joining the plasma membrane NHE3 complex with Ca2+ elevation. Through these studies, a comprehensive description of the formation, function, and fate of this complex will be developed. The mechanisms by which NHE3 is regulated by elevated Ca2+, which will be evaluated, include changes in plasma membrane NHE3 containing complexes, phosphorylation, and rates of trafficking. The proposed studies will increase understanding of (1) mechanisms by which NHE3 is regulated by the second messenger system elevated Ca2+/protein kinase Calpha, (2) NHE3 containing mutt protein plasma membrane complexes involved, and (3) regulatory role of BB PDZ domain containing proteins. The roles in Ca2+ regulation of NHE3 of E3KARP, alpha- actinin-4 and PKCalpha will be examined. The proposal will study NHE3 regulation predominantly in Caco-2 cells and ileal Na+ absorptive cells with some initial screening studies done in PS120 fibroblasts, progressing from molecular studies in cell culture models to relevance of the findings in normal intestinal Na+ absorptive cells.